Replication of the diploid human genome involves a series of complex reactions occurring in at least four phases: initiation of synthesis at origins, elongation on the leading strand, elongation on the lagging strand and replacement of RNA primers with DNA. Current models suggest that more than one DNA polymerase is required for replication, that the proteins that start or finish chains may be different than those that perform the bulk of chain elongation and that the proteins that replicate the leading and lagging strands may be different. Our long-term goal is to define the fine structure of DNA replication fidelity. To do so, we are examining the fidelity of DNA synthesis catalyzed by eukaryotic DNA polymerases alpha, beta, delta and gamma and the fidelity of bidirectional DNA replication by the multiprotein replication apparatus in extracts of human HeLa cells. We have found that polymerases have distinctly different error rates and specificities, which have implications for their roles in the various stages of DNA replication. We have described base substitution and frameshift error rates for the replication apparatus and obtained data suggesting that exonucleolytic proofreading occurs during replication of both the leading and lagging strands. We have found different error rates on the two strands for frameshift errors at two non-reiterated sequence positions. We have begun to examine a large number of errors and template positions using a forward mutation assay. In order to better understand the effects of known mutagens and carcinogens on the fidelity of DNA synthesis, we have also recently initiated studies of replication fidelity with DNA molecules containing defined lesions, including psoralen monoadducts, UV photoproducts and AAF adducts. We intend to continue these studies to define error rates for each of the four phases of replication.